KR100364448B1 - Calcium free subtilisin mutants - Google Patents

Abstract

Novel calcium free subtilisin mutants are taught, in particular subtilisins which have been mutated to eliminate amino acids 75-83 and which retain enzymatic activity and stability. Recombinant methods for producing same and recombinant DNA encoding for such subtilisin mutants are also provided.

Description

Calcium-unbound subtilisin mutant {CALCIUM FREE SUBTILISIN MUTANTS}

(1) Field of invention

The present invention relates to a subtilisin protein modified to remove calcium binding. More specifically, the present invention provides novel subtilisin I-S1 and I-S2 subtilisin mutants, in particular amino acids 75-83, which are deleted from the calcium A-binding loop, and increased heat. It relates to a BPN 'mutation that may further comprise one or more other mutations, such as subtilisin modifications, that restore coordination to stability and / or folding reactions.

(2) Description of related technology

Subtilisin is an exceptional example of a monomeric protein that has a substantial dynamic barrier to folding and unwinding. Its well known example, subtilisin BPN ', is a 275 amino acid serine protease secreted by Bacillus amyloliquefaciens . This enzyme is of great industrial importance and has been the subject of many protein engineering studies [Siezen et al., Protein Engineering, 4: 719-737 (1991); Bryan, Pharmaceutical Biotechnology, 3 (B): 147-181 (1992); Wells et al., Trends Biochem. Sci., 13: 291-297 (1988). Amino acid sequences for subtilisin BPN 'are known in the art and can also be found in the literature (Vasantha et al., J. Bacteriol., 159: 811-819 (1984)). Amino acid sequences found in these documents are cited herein [SEQ ID NO: 1]. When we refer to the amino acid sequence of subtilisin BPN 'or mutants thereof throughout the present specification, we refer to the amino acid sequence cited above.

Subtilisin is a serine protease produced by Gram-positive bacteria or by fungi. The amino acid sequences of many subtilisins are known (Siezen et al., Protein Engineering, 4: 719-737 (1991)). Such are five subtilisin derived from the Bacillus lineage, for example subtilisin BPN ', subtilisin Carlsberg, subtilisin DY, subtilisin amylosacaricitis, and mesentopeptides. I [Vasantha et al., "The genes for alkaline proteases and neutral proteases obtained from Bacillus amyloliquefaciens contain a large open-reading frame between the signal sequence and the region encoding the mature protein", J. Bacteriol., 159: 811-819 (1984); Jacobs et al., “Clone Sequencing and Expression of Subtilisin Carlsberg Obtained from Bacillus rickeniformis,” Nucleic Acids Res. , 13: 8913-8926 (1985); Nedkov et al., "Measurement of the Complete Amino Acid Sequence of Subtilisin DY and Its Comparison with Primary Structures of Subtilisin BPN ', Carlsberg and Amylosacaritis," Biol. Chem. Hoppe-Seyler, 366: 421-430 (1985); Kurihara et al., "Subtilisin Amylosacaritis" J. Biol. Chem., 247: 5619-5631 (1972); And Svendensen et al., "Complete amino acid sequence of alkaline mesentericcopeptidase", FEBS Lett., 196: 228-232 (1986).

The amino acid sequence of the subtilisin obtained from two fungal proteases is known: Proteinase K from Tritirachium albam [Jany et al., "Proteinase K from Trityrachium albam rimber" , Biol. Chem. Hoppe-Seyler, 366: 485-492 (1985)] and thermomycolase from the thermophilic fungus Malbranchea pulchella [Gaucher et al., "Endopeptidase: Thermomime Thermomycolin ", Methods Enzymol., 45: 415-433 (1976).

The enzymes have been shown to be involved in subtilisin BPN 'not only through their primary structure and enzymatic properties, but also by comparison of x-ray crystallographic data [McPhalen et al., "Complex state with subtilisin Carlsberg Crystallization and Molecular Structure of Inhibitor Eglin from Leech FEBS Lett., 188: 55-58 (1985) and Pahler et al., “The three-dimensional structure of the fungal proteinase K shows similarity to bacterial subtilisin. ", EMBO J. , 3: 1311-1314 (1984)].

Subtilisin BPN 'is an example of a particular subtilisin gene secreted by Bacillus amyloliquefaciens. This gene was cloned, sequenced and expressed at high levels from its native promoter sequence in Bacillus subtilis. The structure of subtilisin BPN 'is very dense at 1.3 kV resolution (R = 0.14) and the two ionic binding sites are structurally detailed [Finzel et al., J. Cell. Biochem. Suppl., 10A: 272 (1986); Pantoliano et al., Biochemistry 27: 8311-8317 (1988); McPhalen et al., Biochemistry 27: 6582-6598 (1988). One of them (site A) binds Ca ²⁺ with high affinity and is located near the N-terminus, while the other (site B) binds much weaker to calcium and other cations and is located about 32 Å away. (FIG. 1). Structural evidence for the two calcium binding sites has also been reported by Bod et al. For the homologous enzyme subtilisin Carlsberg [Bode et al., Eur. J. Biochem. , 166: 673-692 (1987).

Further in this respect, the primary calcium binding sites in all subtilisin of groups I-S1 and I-S2 [Siezen et al., 1991, Table 7 below] are nearly identical nine residue loops at the same position of helix C. It is formed from. The X-ray structures of the I-S1 subtilisin BPN 'and Carlsberg, and also the I-S2 subtilisin sabinase, were measured at high resolution. As a result of the comparison of these structures, all three had almost identical calcium A-sites. Has been proved.

Also known are the x-ray structures of the first class of subtilases , namely thermases obtained from Thermoactinomyces vulgaris . Although the overall homology of BPN 'to the thermitase is much lower than the homology of BPN' to the I-S1 and I-S2 subtilisin, the thermase has been found to have similar calcium-A sites. . In the case of the thermitase, the loop is a block of 10 residues at the same site of helix C.

Calcium binding sites are a common feature of extracellular microbial proteases, probably because they contribute significantly to thermodynamic and mechanical stability [Matthews et al., J. Biol. Chem. , 249: 8030-8044 (1974); Voordouw et al., Biochemistry , 15: 3716-3724 (1976); Betzel et al., Protein Engineering , 3: 161-172 (1990); Gros et al., J. Biol. Chem. , 266: 2953-2961 (1991). The thermodynamic and mechanical stability of subtilisin is believed to be essential by the difficulties of the extracellular environment in which subtilisin is secreted, which is filled with protease by its presence. Thus, a high activation barrier to unfolding may be necessary to fix the original form and to prevent transient unfolding and proteolysis.

Unfortunately, the major industrial use of subtilisin is in environments containing high concentrations of metal chelators that deprive calcium from subtilisin and impair its stability. Therefore, it will actually be very important to produce very stable subtilisin independent of calcium.

We have already used several strategies to build a simple thermodynamic model to make it similar to the spreading transition, in order to increase the stability of subtilisin against thermal denaturation [Pantoliano et al., Biochemstry , 26: 2077]. -2082 (1987); Pantoliano et al., Biochemstry , 27: 8311-8317 (1988); Pantoliano et al., B iochemstry , 28: 7205-7213 (1989); Rollence et al., CRC Crit. Rev. Biotechnol. , 8: 217-224 (1988). However, improved subtilisin mutants that are stable in an industrial environment, such as damaging metal chelators and not binding to calcium, are not currently available.

(Object and Summary of the Invention)

It is therefore an object of the present invention to provide modified mutated or modified subtilisin enzymes, such as the subclass I class, to remove calcium binding. As used herein, the term “mutated or modified subtilisin” is meant to include all serine protease enzymes that have been modified to remove calcium bonds. This includes in particular serine proteases which show homology to subtilisin BPN 'and subtilisin BPN', in particular the subclass I class. However, as used herein, and under the definition of mutated or modified subtilisin enzymes, the mutations of the invention are subtilisin BPN ', subtilisin Carlsberg, subtilisin DY, subtilisin amyl It may be considered homologous because it has at least 50%, and preferably 80%, amino acid sequence identity with the above-mentioned sequence for rosakariticus, mesentiopeptidase, thermitase, or savinase Can be incorporated into any serine protease.

Mutated enzymes of the invention are more stable in the presence of metal chelators and may also include increased thermal stability compared to natural or wild type subtilisin. Thermal stability is a good indicator of the overall ROBUSTNESS of the protein. Proteins with high thermal stability are sometimes stable (0), in the presence of a surfactant, and under other conditions that normally deactivate the protein. Therefore, thermally stable proteins are expected to be useful in many industrial and therapeutic applications where resistance to high temperatures, stringent solvent conditions, or extended half-life is required.

Furthermore, a combination of individual stabilizing mutations of subtilisin was found to frequently result in a near cumulative increase in the free energy of stabilization. Thermodynamic stability has also been shown to be related to resistance to irreversible deactivation at high temperatures and high pH. Single site changes of the present invention do not individually exceed 1.5 Kcal / mol in contribution to the free energy of folding. However, this small increase in free energy of stabilization results in a dramatic increase in overall stability when the mutations are combined, because for most proteins the total free energy of folding is in the range of 5 to 15 Kcal / mol. Creighton, Proteins: Structure and Molecular Properties , WH Freeman and Company, New York (1984).

X-ray crystallographic analysis of various combination mutants revealed that the morphological changes associated with each mutation were highly localized with minimal distortion of the skeletal structure. As such, a very large increase in stability can be achieved without a radial change in the tertiary structure of the protein and only by a minor change of the independent amino acid sequence. As set forth prior to the present invention, Holmes et al., J. Mol. Biol. 160: 623 (1982)], the contribution to the free energy of stabilization can be obtained in several ways, including improved hydrogen bonding and hydrophobic interactions in the folded form and reduced chain entropy of the unfolded enzyme. This is important because thermostable enzymes generally have extended half-lives over a wider temperature range, thereby improving performance as a biological reactor and shelf life.

As mentioned above, the present invention provides subtilisin mutants comprising one or more deletion, substitution or addition mutations that provide for removal of calcium binding. Preferably, this will be by deletion, substitution or insertion into the calcium A-site, which in the case of subclass I of class I comprises 9 amino acid residues in helix C. For subtilisin BPN 'the subtilisin mutants will probably comprise one or more addition, deletion or substitution mutations of amino acids at positions 75 to 83 of sequence ID NO: 1, most preferably amino acids 75 to 83 Will include fruitfulness. Deletion of amino acids 75-83 was found to effectively eliminate calcium binding to the resulting subtilisin mutant, while still providing subtilisin BPN 'which still has enzymatic activity.

Such subtilisin mutants without amino acids 75 to 83 of SEQ ID NO: 1 may further comprise one or more amino acid addition mutations in the sequence, such as a mutation that provides reduced proteolysis. It is therefore another object of the present invention to produce subtilisin mutants which have no calcium binding activity and further mutated to return the cooperation to the folding reaction, thereby increasing the stability to proteolysis. Another object of the present invention is to provide thermostable subtilisin mutants that contain a specific combination of mutations that do not bind calcium and provide substantially increased thermal stability.

In particular, the subtilisin mutants of the present invention include subtilisin from the Bacillus strains, such as subtilisin BPN ', subtilisin Carlsberg, subtilisin DY, subtilisin amylosarkariticus and subtilisin There are mesentopeptidase and these include one or more deletion, substitution or addition mutations.

The present invention further provides a subtilisin mutant free of amino acids 75-83 of SEQ ID NO: 1, which results in greatly improved stability by having artificially new protein-protein interactions around the deletion. More specifically, mutations are provided at 10 specific sites in subtilisin BPN 'and analogs thereof, of which 7 have been found to increase the stability of subtilisin proteins individually and in combination. Thus, improved calcium-free subtilisin is provided by the present invention.

(General purpose of the invention)

It is a general object of the present invention to provide subtilisin mutants that are mutated to prevent binding to calcium.

Another object of the present invention is to provide a DNA sequence that provides subtilisin mutants that do not bind calcium upon expression.

Another object of the present invention is to provide subtilisin mutants comprising certain combinations of mutations that provide increased thermal stability.

Another object of the present invention is to provide a method for synthesizing a subtilisin mutant that does not bind calcium by expression in an appropriate recombinant host cell of the subtilisin DNA comprising one or more substitutions, deletions or additions. To provide.

A more specific object of the present invention is to provide a class I subtilase mutant, in particular a BPN 'mutant which is mutated to prevent calcium binding.

Another specific object of the present invention is to provide a DNA sequence which induces a class I subtilase mutant and, in particular, a BPN 'mutant that does not bind calcium.

Another specific object of the present invention is to provide subtilisin I-S1 or I-S2 mutants, and in particular class I subtilase mutant DNA sequences, and more specifically one or more substitutions, in appropriate recombinant host cells. It is to provide a method for producing a BPN 'mutant that does not bind to calcium by expression of a BPN' DNA coding sequence, including an addition or deletion mutation.

Another specific object of the present invention is a special combination of mutant subtilisin I-S1 or I-S2 , and more specifically mutations that do not bind calcium but also provide increased thermal stability or restore cooperativeness to the folding reaction. It is to provide a BPN 'mutant comprising.

The subtilisin mutants of the present invention are intended to be utilized in applications where subtilisin is currently used. If these mutants do not bind to calcium they should be quite suitable for use in an industrial environment, especially including chelating agents, for example surfactant compositions, which substantially reduce the activity of wild type calcium binding subtilisin.

1 is an X-ray crystal structure of S15 subtilisin.

A. The α-carbon plot shows the location of the mutations as is well known. The numbering of wild type subtilisin is maintained. The spherical spheres with dots indicate the position of calcium at the weak ionic binding site (B-site) and the previous position of the high affinity binding site (A-site). There is no A-site loop (crash) in this mutant. N- and C- termini are also indicated. N-terminal is disordered (dotted line).

B. Magnified view of A-site deletion. The loop from the S12 subtilisin is indicated by the dotted line with the continuous helix of s15. Shown in duplicate is the 3 ^* sigma differential electron density (phase from FO12-FO15, S15) representing the deleted A-loop.

2 is an X-ray crystal structure of the calcium A-site region of S12 subtilisin. Calcium is shown as a sphere, represented by a point with half the van der Waals radius. Intermittent lines represent coordination bonds and dotted lines represent hydrogen bonds of 3.2 kPa or less.

3 shows the colorimetric analysis scans of differential scanning calorimetry Apo-S12 (Tm = 63.5 ° C.) and S15 (Tm = 63.0 ° C.). Measurements were performed using a Hart 7707 DSC (Differential Scanning Calorimetry) thermally conductive scanning microcolorimeter (Pantoliano et al., Biochemstry , 28: 7205-7213 (1989)). Sample conditions were 50 mM glycine, pH 9.63, scan rate 0.5 ° C./min. Excess heat capacity is measured in μJ / °. Colorimetric ampoules contain 1.78 mg of protein.

4 is a colorimetric analysis of subtilisin S11. The heat of calcium binding to the continuous addition of calcium is expressed as the ratio of [Ca] / [P]. The data given best match the binding curve calculated using the equation (1) in the description below, assuming a coupling constant of ΔH and 7 × 10 ⁶ equal to 11.3 kcal / mol. In this titration, [P] = 100 μΜ and the temperature was 25 ° C.

5 shows the kinetics of calcium breakdown from subtilisin S11 as a function of temperature. 1 μM subtilisin S11 was added to 10 μM Quin2 at time zero. Calcium breaks down from subtilisin and binds to Quin 2 until a new equilibrium is achieved. Calcium degradation rate is followed by an increase in fluorescence of Quin 2 when Quin 2 binds to calcium.

A. The log of% of protein bound to calcium is plotted against time. Decomposition kinetics at four temperatures is shown. Decomposition follows the primary kinetics for the first 25% of the reaction. This is so until equilibrium is reached, so calcium recombination can be ignored.

B. Temperature dependence of the rate at which calcium decomposes from S15 in the presence of excess Quin 2, pH 7.4 at temperatures ranging from 25 to 45 ° C. A graph of the natural logarithm of the equilibrium constant for the transition state (calculated from the aering equation) versus the absolute temperature is shown. The line fits according to equation (3) T ₀ = 298 K in the description below.

6 Analysis of subtilisin refolding monitored by circular dichroism (CD).

A. The CD spectrum is shown for S15 as follows: (1) S15 in 25 mM H ₃ PO _{4 at} pH 1.85; (2) S15 neutralized at pH 1.85 and then neutralized to pH 7.5 by adding NaOH; (3) S15 neutralized to pH 7.5 by addition of KCl to 0.6 M after 30 minutes of denaturation at pH 1.85; And (4) natural S15 subtilisin. The protein concentration in all samples was 1 μM.

B. Mechanics of S15 refolding. The sample was denatured at pH 1.85 and then the pH was adjusted to 7.5. At time zero KCl was added to the denatured protein. Recovery of the intrinsic structure occurred at 222 nm at KCl concentrations of 0.3 and 0.6 M. A 0.6 M sample 30 minutes after refolding was used to record the corresponding spectrum of A above.

7 shows the kinetics of refolding of S15 as a function of ionic strength.

A. The log of% of unfolded protein is shown in a time zone graph. The mechanics of refolding are shown at four ionic strengths. The amount of refolding was measured by circular dichroism (CD) from an increase in negative ellipticity at 222 nm. 100% fold is determined from the signal at 222 nm for native S15 at the same concentration and 0% fold is measured from the signal for acid-modified S15. Refolding approximately follows the primary dynamics for the first 90% of the response. Refolding was performed at 25 ° C.

B. The logarithm of the first order rate constant for refolding obtained by CD or fluorescence measurement at 25 ° C. is plotted as a function of the logarithm of ionic strength. Ionic strength varied from I to about 0.25 to I = 1.5. The refold speed increases linearly with log I. A 10-fold increase in I results in an approximately 90-fold increase in refold speed.

FIG. 8 is the temperature dependence of the refold rate of S15 subtilisin in 0.6 M KCl, 23 nM KPO ₄ pH 7.3. The natural logarithm of the equilibrium for the transition state (calculated from the aering equation) is shown as the inverse of the absolute temperature versus the graph. The line fits well with the equation (3) T _O = 298 K in the description below.

9 is an X-ray crystal structure of weak ionic bond of S15 subtilisin. Coordination bonds are shown in dashed lines. Note the preponderance of charged amino acids.

(Explanation of preferred embodiment)

As discussed above, calcium binding contributes substantially to the thermodynamic and mechanical stability of extracellular microbial proteases. Moreover, with regard to subtilisin, a high activation barrier to annealing is given a protease-filled environment, which preserves its native form and is the place where transient annealing and subtilisin are secreted, and consequently proteolysis when autolysis occurs. It may be necessary to prevent. The release reaction of subtilisin can be divided into two parts as follows:

N (Ca) ⇔ N ⇔ U

Where N (Ca) is the native form of subtilisin in which calcium is bound to the high affinity calcium-binding site A [Finzel et al., J. Cell. Biochem. Suppl. , 10A: 272 (1986); Pantoliano et al., Biochemistry , 27: 8311-8317 (1988); McPhalen et al., Biochemistry , 27: 6582-6598 (1988); N is a folded protein without bound calcium; U is an unwrapped protein. Subtilisin is a relatively stable protein whose stability is mostly mediated by high affinity calcium sites [Vororwöw et al., Biochemistry , 15: 3716-3824 (1976); Pantoliano et al., Biochemistry , 27: 8311-8317 (1988). The melting point temperature of subtilisin at pH 8.0 is approximately 75 ° C. in the presence of micromolar calcium and approximately 56 ° C. in the presence of excess EDTA [Takehashi et al., Biochemistry , 20: 6185-6190 (1981); Bryan et al., Prac. Natl. Acad. Sci. USA , 83: 3743-3745 (1986b). Previous colorimetric analysis of the calcium-non-binding (apoenzyme, or protein part of the enzyme) form of subtilisin indicated that it was boundary stability at 25 ° C. at which time ΔG of annealing was less than 5 kcal / mol [Pantoliano et al., Biochemistry , 28: 7205-7213 (1989). Since calcium is an integral part of the subtilisin structure, apoenzymes are believed to be intermediates in the folded state of subtilisin.

In order to independently investigate the two phases of the folding process, we constructed a series of mutant subtilisins. First, all proteolytic activity was investigated to prevent autolysis occurring during unfolding and refolding reactions. This can be done, for example, by converting the active site serine 221 to cysteine. [S221A mutants were originally constructed for this purpose. The mature form of this mutant is heterologous on its N-terminus, but this is probably due to some inaccurate processing of the pro-enzyme. This mutation shows little effect on the heat denaturing temperature of subtilisin, but Reduce the peptidase activity of subtilisin by a factor of 3 × 10 ⁴ (Abrahmsen et al., Biochemistry , 30: 4151-4159 (1991)). This mutant therefore enables the folding of the subtilisin to be studied without complicating proteolysis. In the present invention, the notation for indicating an amino acid substitution uses a single letter amino acid code of an amino acid to be substituted after a number indicating where the substitution is to be made in the amino acid sequence, and after a single letter of the amino acid to be inserted therein. For example, S221C indicates that serine 221 is substituted with cysteine. Subtilisin mutants in which such a single amino acid is substituted are represented by subtilisin S221C. The resulting S221C subtilisin mutant is represented by S1.

Subtilisin may further be mutated to make relatively unstable apoenzymes to make preparation and purification easier. Versions of S1 comprising 3 or 4 additional amino acid mutations, for example M50F, Q206I, Y217K and N218S, can also be used in the methods of the invention. Such additional mutations cumulatively increase the free energy of annealing by 3.0 kcal / mol and increase the thermal denaturation temperature of the apoenzyme by 11.5 ° C. [Pantoliano et al., Biochemistry , 28: 7205-7213 (1989)]. . Mutants containing M50F, Q206I, Y217K, N218S and S221C are denoted S11, and mutants containing M50F, Y217K, N218S and S221C are denoted S12. [The specific activities of S11 and S12 and S15 on the synthetic substrate SAAPFna are identical (SA is about 0.0024 U / mg at 25 ° C., pH 8.0). This measurement was performed on freshly purified protein on mercury affinity column.]

In order to prepare a subtilisin BPN 'protein lacking calcium binding activity, the inventors chose to delete the binding loop at the calcium A-site to artificially manipulate the novel calcium-unbound subtilisin protein. This loop contains an obstruction to the subtilisin BPN 'α-helix comprising amino acids 63-85 of SEQ ID NO: 1 [McPhalen and James, 1988]. Residues 75-83 of the subtilisin BPN 'protein form a loop that prevents the last rotation of the alpha helix of 14 residues comprising amino acids 63-85 [SEQ ID NO: 1]. [This set of 9 residues was chosen for deletion as opposed to 74-82 (these residues actually belong to the loop) excluding Ala 74 rather than Gly 83 in the resulting successive helix. Alanine is statistically more likely to be present in the α-helix because of its broader, accessible framework of glycine.] The four carbonyl oxygen ligands for calcium are amino acids 75-83 [SEQ ID NO: 1]. Provided by a loop that is configured. The geometry of a ligand is a pentagonal 2-pyramid whose axis passes through the carbonyls of amino acids 75 and 79. On one side of the loop is a carboxylate (D41) with two projections of this shape, on the other side the N-terminus of the protein and the side chain of Q2. The seven coordination distances range from 2.3 to 2.6 mm 3, with the shortest belonging to aspartyl carboxylate. Three hydrogen bonds connect the N-terminal fragments in a beta configuration parallel to the loop residues 78-82. High affinity calcium binding sites are a common feature of subtilisins that make a significant contribution to their high stability. By binding at specific sites in the subtilisin tertiary structure, calcium provides its binding free energy to the stability of the intrinsic state. In the present invention, site-specific mutagenesis was used to delete amino acids 75-83 of SEQ ID NO: 1, thereby creating an uninterrupted helix and eliminating the possibility of calcium binding at site A (FIGS. 1A and 1B). .

The inventors believed that a stabilization strategy based on calcium binding could enable survival in an extracellular environment. Since the main industrial use of subtilisin is in an environment containing high concentrations of metal chelators, for the present inventors it is indeed very important to produce stable subtilisin, which is independent of calcium and thus unaffected by the presence of metal chelating agents. It was.

We chose to remove calcium bonds by removing these amino acids, while replacing one or more amino acids at positions 75-83 with other mutations, eg, replacement amino acids, It should be possible to remove calcium bonds even by insertion, substitution and / or deletion. This can also be done by site-directed mutagenesis.

Thus, since this loop is common to subtilisin, equivalent mutations to other subtilisin, especially class I subtilisin, such as by site-specific mutagenesis, likewise remove calcium binding and are enzymatic Will provide an active mutant.

In particular, we synthesized three subtilisin BPN 'DNAs that were mutated to further remove amino acids 75-83 involved in calcium binding by site-specific mutagenesis and further comprising additional substitution mutations. These mutated subtilisin BPN 'DNAs provide subtilisin proteins that have increased thermal stability upon expression of DNA and / or are resistant to proteolysis.

Specific subtilisin BPN 'mutants synthesized by the inventors are denoted in this application as S15, S39, S46, S47, S68, S73, S79 and S86. Specific point mutations described herein identify specific amino acids of the subtilisin BPN 'amino acid sequence, as described by SEQ ID NO: 1, and are mutated in accordance with the present invention. For example, the S15 mutant comprises deletions of amino acids 75-83 and further includes the following substitution mutations: S221C, N218S, M50F and Y217K. S39 mutants similarly include deletions of amino acids 75-83 and further include the following substitution mutations: S221C, P5A, N218S, M50F and Y217K. S46 mutants include deletions of amino acids 75-83 and further include the following substitution mutations: M50F, Y217K and N218S. S47 mutants include deletions of amino acids 75-83 and further include the following substitution mutations: P5A, N218S, M50F and Y217K. S68 mutants include deletions of amino acids 75-83 and further include the following substitution mutations: P5S, N218S, M50F and Y217K. S73 mutants include deletions of amino acids 75-83 as well as the following substitution mutations: Q2K, M50F, A73L, Q206V, Y217K and N218S. S79 mutants include deletions of amino acids 75-83 and further include the following substitution mutations: Q2K, M50F, A73L, Q206C, Y217K and N218S. Finally, S86 mutants include deletions of amino acids 75-83 as well as the following substitution mutations: Q2K, S3C, M50F, A73L, Q206C, Y217K and N218S. The specific activity of proteolytically active S46, S47, S68, S73, S79 and S86 subtilisin was found to be similar or increased compared to wild type enzymes.

Various Δ75-83 (meaning that the amino acids 75-83 are deleted) subtilisin synthesized by the inventors are listed in Table 1 below. Particular points of mutations that occurred in the amino acid sequence of the subtilisin BPN 'amino acid sequence are shown, as described in SEQ ID NO: 1. The synthesis of these mutants is described in more detail below.

Subtilisin mutation S221C P5A △ 75-83 N218S M50F Q206I Y217K Q271E Q2K A73L Q206V Q206C S3C BPN ' - - - - - - - - - - - - - S1 ^* + - - - - - - - - - - - - S11 ^* + - - + + + + - - - - - - S12 ^* + - - + + - + - - - - - - S15 ^* + - + + + - + - - - - - - S39 + + + + + - + - - - - - - S46 - - + + + - + - - - - - - S47 - + + + + - + - - - - - - S68 - P5S + + + - + - - - - - - S73 - - + + + - + + + + + - - S79 - - + + + - + + + + - + - S86 - - + + + - + + + + - + +

The plus sign in the table indicates that the subtilisin contains a special mutation. The X-ray crystal structure of wild type, S12 and S15 was determined to be 1.8 mm 3.

* S1, S11, S12, S15 and S39 are slow activity mutants that are configured to help assess structural and morphological stability.

To understand how calcium binding contributes to the stability of subtilisin, the thermodynamics and dynamics of calcium binding to high affinity calcium A-sites were measured by microcalorimeters and fluorescence spectrometers. Calcium-binding is a process in which the binding constant (K _a) driven by enthalpy, such as 7 × 10 ⁶ M ^-1. The dynamic barrier (23 kcal / mol) for calcium removal from the A-site is substantially greater than the standard free energy of binding (9.3 kcal / mol). The kinetics of calcium breakdown from subtilisin (such as in excess EDTA) are therefore very slow. For example, the half-life (t _1/2 ) at which calcium from subtilisin is decomposed, such as half the time at half calcium of subtilisin, is 1.3 hours at 25 ° C.

X-ray crystallography shows that the structures of the subtilisin mutant and wild type are very similar, except for the deleted calcium-binding loop region. The N-terminus of the wild-type protein lies next to the site A loop and provides one calcium coordination ligand, the side chain oxygen of Q2. In Δ75-83 subtilisin, residues 1 to 4 become disordered when the loop is removed. These first four residues are disordered within the X-ray structure because all of their interactions are with the calcium loop. N-terminal sequencing confirms that the first four amino acids are present, confirming that processing occurs at the normal site. The spiral appears to be unobstructed and shows normal spiral geometry over its entire length. X-ray crystallography also shows that the structure of subtilisin with and without deletions overlaps with a mean square difference (rms) of 0.17 Hz between 261 α-carbon positions, which, given the size of the deletions, Remarkably similar. However, the diffusion difference density and higher temperature factors indicate that the newly exposed residues adjacent to the deletion site are slightly disordered.

While removal of calcium bonds is beneficial because it produces more stable proteins even in the presence of metal chelating agents, it is disadvantageous in at least one aspect. Specifically, removing the calcium loops without any other offsetting mutations causes destabilization of the original state with respect to the partially folded state, thus losing the morphology of the folding. We therefore sought to genetically engineer the subtilisin S15 BPN 'protein deficient in 75 to 83 amino acids to restore coordination to the folding reaction. In the best designed proteins, all parts of the molecule are interdependent, thereby making the annealing reaction highly cooperative. The coordination of the folding reaction makes it possible to achieve sufficient stability of the original state for the proper purpose, since the overall stability of the original form is approximately equal to the sum of all local interactions.

Therefore, while Δ75-83 subtilisin is an example of an active and stable engineered subtilisin that is active in the absence of calcium, we sought to improve this protein by further mutations. The design of especially highly stable calcium-unbound subtilisin relies on repeated engineering cycles. We have found that the essential first step in the cycle is to significantly reduce the proteolytic activity of subtilisin. This is essential because calcium contributes greatly to the morphological stability of subtilisin and the initial conversion of calcium-unbound subtilisin is sensitive to proteolytic degradation. After reducing the sensitivity to proteolysis, the next step in the cycle was to remove the sequences necessary for calcium binding, namely A-sequence. Although the S15 Δ75-83 subtilisin is much less stable than the wild type subtilisin in the presence of calcium, this mutant is more stable than the wild type subtilisin in the presence of the metal chelator EDTA.

Thus, the third step was to improve the stability of the calcium-unbound subtilisin protein. In order to improve the stability of the calcium-free subtilisin, we then sought to create a home of disordered N-terminal residues. To produce highly stable calcium-unbound subtilisin, the N-terminal portion of the destabilized protein may be modified by deletion of the calcium A-loop. For example, the disordered N-terminus can be deleted or extended. However, this is somewhat problematic because it is not known what is needed to process propeptides from mature proteins. Therefore, it is known that processing sites are not determined by amino acid sequences because the mutants Y1A (C-terminus of the propeptide), A1C and Q2R do not alter the cleavage site. It is also known that the original structure of the N-terminus of subtilisin does not measure cleavage sites because the Δ75-83 variant is processed correctly. Since it is not yet known how to change the processing site, the interaction with the existing N-terminus can be optimized.

Investigations into the structure of S15 subtilisin have shown a number of possibilities for improving the stability of mutant enzymes. The regions most affected by deletions are the N-terminal amino acids 1-8, 36-45 ω-loop, 70-74 α-helix, 84-89 helix rotation and 202-219 β-ribbon. As previously described, the first four residues in the Δ75-83 subtilisin are disordered in the X-ray structure since all of its interactions are with the calcium loop. However, N-terminal sequencing shows that the first four amino acids are present, which confirms that processing occurs at the normal site. Besides the N-terminus, there are three other residues whose side chain form is clearly different from the wild type. Y6 is an indirect effect of destabilization at the N-terminus and redirects from the surface location to the position where more solvent is exposed. The precursor calcium ligands, D41 and Y214, are in coordination order to form new hydrogen bonds. All three residues significantly increase in B-factor due to the deletion of amino acids 75-83. In addition, S87 and A88 do not change form but show significantly increased B-factors. P86 terminates the α-helices from which the calcium loop is deleted. In view of the above, other mutations in one or more of the above-mentioned sites, or in amino acids adjacent thereto will provide subtilisin BPN ′ with greater enzymatic activity or increased stability.

There are several logical strategies for remodeling this region of the protein to produce subtilisin BPN 'mutants with greater enzymatic activity or increased stability. Since the four amino acids at the N-terminus are disordered in the X-ray structure, one possible approach would be to delete them from the protein. However, the requirements for processing propeptide from mature proteins are currently unknown. Deletion of amino acids from the insertion or N-terminal region is therefore problematic. For this reason, insertions or deletions in the N-terminal region are advantageously avoided for amino acid substitutions. It can be assumed that many of the original amino acids in the above-described regions of subtilisin that interact with amino acid 75-83 loops are no longer optimal. Therefore, it was possible to increase the stability of the molecule by substitution, deletion or addition of at least one amino acid at a location where its environment was changed by 75-83 deletion.

The first attempt was to mutate proline to alanine at position 5 to make the flexibility at position 5 even greater. This increased flexibility allows the N-terminus to make an effort to find unique locations along the new surface of the protein produced by the deletion of the calcium loop. Once the N-terminus estimates a unique location, its local interaction can then be optimized.

P5A mutations have been made in an effort to create greater flexibility for the N-terminus and make it possible to find unique locations along new surfaces of proteins produced by the deletion of calcium loops. In the original structure, the first five amino acids take an extended form and form β-pair hydrogen bonds with calcium loops as well as Q2 side chain interactions with calcium. Proline at position 5, conserved among seven bacterial subtilisins with homologous calcium A-sites, may help stabilize the extended form. The P5A mutation in Δ75-83 subtilisin should therefore lead to an increase in the cooperativeness of the annealing response. The X-ray structure of this variant was measured at 1.8 Hz.

We have selected amino acids at ten different positions where the environment has changed substantially for substitution. Mutation generation and screening procedures have been developed to screen all possible substitutions at specific sites. Techniques for generating and screening subtilisin variants include mutagenesis of cloned subtilisin genes in vitro, expressing the mutated genes in Bacillus subtilis, and screening for increased stability.

For example, site-specific mutagenesis was performed on the S46 subtilisin gene using oligonucleotides degenerate in one codon. The degenerate codon used all combinations of the sequence NNB, where N is any one of the four nucleotides and B is T, C or G. The 48 codons represented in this population code for all 20 amino acids, except for the ocher and umber termination codons. Mutated genes were used to transform Bacillus subtilis. Examples of specific mutations are shown in Table 2 as follows:

In order to have a 98% chance of finding tryptophan, glutamine, glutamate or methionine in the mutant population, one skilled in the art should screen about 200 mutant clones. Each such codon is represented by only one codon of the 48 codons contained in the population of sequence NNB. Codons for all other amino acids will be required to screen about 100 mutant clones to have a 98% chance that they are represented by at least two codons in the population and represented by the mutant population.

To identify the optimal amino acid at one position, mutants were screened for retention of enzymatic activity at high temperatures. 100 μl of medium was dispensed into each of the 96 wells of the microtiter dish. Each well was inoculated with a Bacillus transformant and incubated with shaking at 37 ° C. After 18 hours of growth, 20 μl of the culture was diluted with 80 μl of 100 mM Tris-HCl, pH 8.0 in a second microtiter dish. This dish was then incubated at 65 ° C. for one hour. The dish was incubated at high temperature then cooled to room temperature and 100 μl of 1 mM SAAPF-pNA was added to each well. The wells that cut the pNA fastest (changing yellow) were considered to contain the most heat-resistant subtilisin mutant. Once preliminary identification of stable mutants was made from the second microtiter dish, the Bacillus clone of the corresponding well of the first microtiter dish was grown for further analysis.

The screening process was found to stabilize the mutation at 7 of 10 positions. As noted, these amino acid positions were chosen at the position of the protein whose environment is substantially changed by the deletion of the calcium domain. No mutations were identified at positions 4, 74 and 214, which themselves increased significantly the half-life of the mutant in connection with the present invention. However, at position 214 only the effect of hydrophobic amino acids was screened. Mutations were not seen at positions 5, 41 and 43, which could be measured but resulted in a low level of increase in stability. Moreover, several mutations were found at positions 2, 3, 73 and 206, which significantly increased the half-life of the mutant relative to the original subtilisin. These stabilizing mutants are shown in Table 3 below:

Stabilization mutation Protein region part increase N-terminal: Q2K 2.0 times 63-85 α-helix: A73L 2.6 times 202-220 β-ribbon Q206V 4.5 times N-terminal-β-ribbon S3C-Q206C (disulfide) 14 times

Subtilisin S73 was then made by combining the stabilizing amino acid modifications at positions 2 (K), 73 (L) and 206 (V). The properties of S73 subtilisin and the properties of S46, S79 and S86 are shown in Table 4 below.

Mutant Mutant ¹ Inactive ² Half-life (60 ℃) ³ increase S46 - 100 U / mg 2.3 minutes - S73 Q2KA73LQ206V 160 U / mg 25 mins 11 times S79 Q2KA73LQ206C ND ⁴ 18 mins 8 times S86 Q2KS3C ⁵ A73LQ206C ⁵ 85 U / mg 80 minutes 35 times 1) Subtilisin S46, S73, S79 and S86 all contain the mutations M50F, Y217K and N218S and Q271E. 2) Inactivation in succinyl-L-Ala- in 10 mM Tris-HCl, pH 8.0 and at 25 ° C. L-Ala-L-Pro-L-Phe-p-nitroanilide (SAAPF-pNA) is measured. 3) The half-life is 60 ° C. in 10 mM Tris-HCl, pH 8.0, 50 mM NaCl, 10 mM EDTA. 4) Not measured. 5) Disulfide bonds were formed between cysteines between positions 3 and 206. The formation of disulfide bonds was confirmed by measuring the radius of rotational motion of the denatured protein by gel electrophoresis.

In many cases, the choice of amino acid at a particular position will be influenced by the amino acid at neighboring positions. Therefore, to find the best combination of stabilizing amino acids, in some cases it will be necessary to change the amino acids at two or more positions simultaneously. In particular, this has been done at positions 3 and 206 where their side chains have amino acids that can interact strongly. It was determined that the best combination of strains was the cysteine strain at positions 3 and 206. This variant is indicated as S86. Because of the proper geometry and close closeness between the cysteines at these two positions, disulfide crosslinks spontaneously form between these two residues.

The stability of S86 subtilisin was studied in relation to S73. The half-life of S86 was found to be 80 minutes at 60 ° C. and in 10 mM Tris-HCl, pH 8.0, 50 mM NaCl and 10 mM EDTA, which is a 3.2 fold increase compared to S73 subtilisin. In contrast, 3-206 disulfide crosslinks would not be formed in natural subtilisin containing calcium A-sites. This is because the 75-83 binding loop separates the N-terminal amino acid from the 202-219 β-ribbon. Therefore, the increase in stability that occurs in the S86 mutants of the invention without the 75-83 binding loop will not be equally observed in native subtilisin that is similarly cysteine modified at these positions.

A similar increase in stability is expected to be unique to other subtilisins of the I-S1 and I-S2 groups if their calcium loops are deleted [Siezen et al., Protein Engineering , 4, pp. 719-737, FIG. 7]. This is a reasonable prediction based on the fact that the primary calcium sites in these different subtilisins are formed from nearly identical nine residue loops constructed at the same position in helix C.

The X-ray structures of I-S1 subtilisin BPN and Carlsberg, as well as I-S2 subtilisin (savinase), were measured at high resolution. Comparing these structures demonstrated that all three had nearly identical calcium A-sites.

Also known are the X-ray structures of the thermase from Thermoactinomyces vulgaris, a subclass of class I. Although the overall homology of BPN 'to the thermitase is much lower than the homology of BPN' to I-S1 and I-S2, it has been found that the thermase has a similar calcium A-site. In the case of the thermitase, the loop is a block of 10 residues at the same site of helix C.

Therefore, the stabilizing mutations exemplified herein provide a similar beneficial effect on the stability of calcium loop-deleting plates of other class I subtilases.

The stability of S73, S76 and S86 in relation to S46 subtilisin was compared by measuring resistance to thermal inactivation at 60 ° C. and in 10 mM Tris-HCl, pH 8.0, 50 mM NaCl and 10 mM EDTA. A constant amount was taken at intervals and then the activity remaining in each constant amount was measured. Under these conditions, the half-life of S46 subtilisin was 2.3 minutes and the half-life of S73 was 25 minutes (see Table 4).

In order to identify other mutants with increased stability, any mutagenesis technique known to those skilled in the art can be used. One example of such a technique for generating and screening subtilisin variants includes the following three steps: 1) in vitro mutagenesis of the cloned subtilisin gene; 2) expression of mutated genes in Bacillus subtilis; And 3) screening for increased stability. The most important element in the random mutagenesis approach is the ability to screen the majority of variants.

Although random mutagenesis can be used, the mutagenesis process described above enables mutations to be specific to localized regions of the protein (eg, N-terminal regions). As noted above, the S46, S47, S68, S73, S79 and S86 mutants (which include the active-site S221) were found to be enzymatically active. It is anticipated that other substitutions may be found to provide equivalent or even greater stability and activity.

The activity of exemplary calcium-unbound subtilisin mutants of the invention on substrate sAAPF-pNA at Tris-HCl, pH 8.0 and 25 ° C. is shown in Table 5 below:

Subtilisin Inactive Half-life (55 ℃) BPN ' 80 U / mg 2 mins S12 0.0025 U / mg ND ¹ S15 0.0025 U / mg ND ¹ S39 0.0025 U / mg ND ¹ S46 125 U / mg 22 mins S47 90 U / mg 4.7 min S68 ~ 100 U / mg 25 mins 1) Half-life was not measured for inactive subtilisin.

As indicated above, subtilisin mutants S46, S47, S68, S73, S79 and S86 had increased catalytic activity compared to subtilisin BPN '. The change in catalytic effect due to deletion was unexpected due to the fact that the active site of subtilisin was spatially separated from the calcium A-site.

The stability of these mutant subtilisin was compared by measuring native subtilisin BPN 'and their resistance to thermal inactivation. Since the stability of the calcium-unbound subtilisin mutants should not be affected by the metal chelating agent, the experiment showed that 10 mM Tris-HCl, pH 8.0, 50 mM NaCl and 10 mM EDTA (EDTA for calcium The binding constant of was performed in 2 x 10 ⁸ M ^-1 ). Protein was dissolved in the buffer and heated to 55 ° C. A constant amount was taken at time intervals to measure the activity remaining in each constant amount. The dynamics of deactivation are plotted in FIG. 10. Under these conditions, the half-life of subtilisin mutants was much improved than that of subtilisin BPN '. These results indicate that subtilisin mutated to remove calcium binding at site A has complete catalytic activity and improved stability in EDTA compared to subtilisin BPN '. Valid stability levels of S46 were achieved without additional mutations that compensated for the loss of interaction caused by deleting amino acids 75-83.

As such, we provided reliable evidence that subtilisin mutants that retain activity and still do not bind calcium can be obtained. Therefore, it is expected that these mutants may be used in industrial environments that include chelating agents. While this only appears to be specific for subtilisin BPN ', equivalent mutations are different serine proteases, most particularly other I-S1 or I-S2 subs, provided they have substantial sequence similarity, especially at calcium binding sites. So should it be with tilisin.

Such a strategy compares the sequence of subtilisin BPN 'for other serine proteases, for example to identify amino acids that are presumed to be required for calcium binding, and then makes appropriate modifications, such as by site-specific mutagenesis. It will involve creating a. Because many subtilisins are involved in subtilisin BPN 'not only through their primary sequence and enzymatic properties, but also by X-ray crystallographic data, other active subtilisin mutants lacking calcium binding are site specific. It is expected that it can be produced by enemy mutagenesis. For example, there is structural evidence that the homologous enzyme subtilisin Carlsberg also includes two calcium binding sites. Similarly, the X-ray structure of the thermitase is known and this subtilisin has a calcium A binding site similar to that of subtilisin BPN '. In the case of the thermitase, the calcium binding loop is an obstacle consisting of 10 residues at the same site of helix C. Therefore, these enzymes must also follow the mutations described herein, which remove calcium binding sites and produce stable and active enzymes. Furthermore, as discussed above, it is demonstrated that the primary calcium binding sites of all subtilisin of the [Siezen et al] I-S1 and I-S2 groups are formed from a loop consisting of nearly identical 9 residues at the same position of helix C. It became. As such, in view of the nearly identical structure of the calcium A-site, the methods described herein should be applied to that not all subtilisins belong to the I-S1 and I-S2 groups described in the literature, such as season.

Or alternatively, if amino acids with calcium binding sites are already known for a particular subtilisin, the corresponding DNA may be deleted to delete one or more such amino acids, or to replace, delete or add mutations that remove calcium binding. Will be mutated by site-specific mutagenesis to provide.

Mutant subtilisin of the invention will generally be produced by recombinant methods, in particular by expression of subtilisin DNA mutated to produce a subtilisin protein that is enzymatically active at expression and does not bind calcium.

Preferably, the subtilisin DNA will be expressed in microbial host cells, in particular Bacillus subtilis. Because the bacterium originally produces subtilisin, effectively secretes proteins, and can produce proteins in active form. However, the present invention is not limited to the expression of subtilisin in basil, but rather includes expression in any host cell that provides for the expression of the desired subtilisin mutants. Suitable host cells for expression are well known in the art and include, for example, bacterial host cells such as E. coli, Bacillus, Salmonella, Pseudomonas; Yeast cells, such as Saccharomyces cerevisiae, Pichia pastoris , Kluveromyces , Candida, Shizuka caromyces ; And mammalian host cells such as CHO cells. However, bacterial host cells are preferred host cells for expression.

Expression of subtilisin DNA will be provided using available vectors and regulatory sequences. The actual choice will depend largely on the particular host cell used for expression. For example, if subtilisin mutant DNA is expressed in Bacillus, Bacillus promoters as well as Bacillus derived vectors will generally be utilized. We particularly used pUB110-based expression vectors and native promoters from the subtilisin BPN 'gene to regulate expression on Bacillus subtilis.

It is further noted that once the amino acid sequence of a particular subtilisin mutant that does not bind calcium is found, it may be possible to prepare the subtilisin mutant by protein synthesis, such as by Merrifield synthesis. However, expression of subtilisin mutants in microbial host cells is generally preferred. This is because it enables microbial host cells to produce subtilisin protein in a form suitable for enzymatic activity. However, as we further teach herein methods for obtaining in vitro refolding of subtilisin mutants, it should be possible to convert improperly folded subtilisin mutants into active form.

The following specific examples are provided to further illustrate the present invention and its advantages, and it is to be understood that the following examples are for illustrative purposes only and not limitation.

Example 1

Cloning and Expression: The subtilisin gene (subtilisin BPN ') from Bacillus amyloliquefaciens was cloned, sequenced and expressed at high levels from its natural promoter sequence in Bacillus subtilis [Wells et al., Nucleic Acids Res. 11: 7911-7925 (1983); Vasantha et al., J. Bacteriol. , 159: 811-819 (1984). All mutant genes were recloned into pUB110-based expression plasmids and used to transform Bacillus subtilis. Bacillus strains used as hosts contain chromosomal deletions of its subtilisin gene and therefore do not produce background wild type (wt) activity [Fahnestock et al., Appl. Environ. Microbial. , 53: 379-384 (1987). Oligonucleotide mutagenesis was performed as previously described [Zoller et al., Methods Enzymol. , 100: 468-500 (1983); Bryan et al., Proc. Natl. Acad. Sci. , 83: 3743-3745 (1986b)]. S221C was expressed at a level of approximately 100 mg of accurately processed mature form per liter in a 1.5 liter New Brunswick Fermentor. The addition of wild-type subtilisin to promote the production of mature forms of S221C subtilisin is not required for the Bacillus host strain of the present invention, unlike in the case of the previous strains [Abrahmsen et al., Biochemistry 30: 4151-4159 (1991). )].

Protein Purification and Characterization: Wild-type subtilisin and mutant enzymes were purified essentially as described in the literature and confirmed for homology [Bryan et al., Proc. Natl. Acad. Sci. 83: 3743-3745 (1986b); pantoliano et al., Biochemistry , 26: 2077-2082 (1987); And Biochemistry , 27: 8311-8317 (1988). In some cases, C221 mutant subtilisin was purified again on sulfhydryl specific mercury affinity column (Affi-gel 501, Biorad). Assays of peptidase activity are described by succinyl- (L) -Ala- (L) -Ala- (L) -Pro- (L) -Phe-p-nitroanilide (hereinafter referred to as sAAPFna), as described by Delmar et al. (See Delmar et al., Anal. Biochem. , 99: 316-329 (1979). Protein concentration [P] was measured using P ^0.1% = 1.17 at 280 nm [Pantoliano et al., Biochemistry , 28: 7205-7213 (1989)]. For variants containing Y217K, ^0.1% P at 280 nm was calculated as 1.15 (or 0.96 X wt) based on the loss of one Tyr residue [Pantoliano et al., Biochemistry , 28: 7205-7213 (1989) ].

N-terminal analysis: The first five amino acids of subtilisin S15 were measured by sequential Edman digestion and HPLC analysis. The results indicated that 100% of the material had the amino acid sequence expected from the DNA sequence of the gene, and the processing of the pro-peptide was done at the same position in the sequence for the mutant as for the wild type enzyme.

(Example 2)

Structure of the calcium A site of S12 subtilisin: The calcium at site A is coordinated by five carbonyl oxygen ligands and one aspartic acid. Four of the carbonyl oxygen ligands for calcium are provided by a loop consisting of amino acids 75-83 (see FIG. 2). The geometry of a ligand is the same as that of a pentagonal 2-pyramid whose axis passes through the carbonyls of 75 and 79. On one side of the loop is the carboxylate (D41), which has two projections of this shape, while on the other side the N-terminus of the protein and the side chain of Q2. The seven coordination distances range from 2.3 to 2.6 mm 3, with the shortest aspartyl carboxylate. Three hydrogen bonds connect the N-terminal fragments in a beta configuration parallel to the loop residues 78-82.

Preparation of Apo-Subtilisin: S11 and S12 subtilisin contain the same molar amount of correctly bound calcium after purification. X-ray crystallography revealed that this calcium is bound to the A site [Finzel et al., J. Cell. Biochem. Suppl. , 10A: 272 (1986); Pantoliano et al., Biochemistry , 27: 8311-8317 (1988); McPhalen et al., Biochemistry , 27: 6582-6598 (1988).

Complete removal of calcium from subtilisin is very slow, requiring dialysis against EDTA for 24 hours at 25 ° C. to remove all calcium from the protein, and then high salt at 4 ° C. to remove all EDTA from the protein. Dialysis for 48 hours is required (Brown et al., Biochemistry , 16: 3883-3896 (1977)). To prepare calcium-unbound forms of subtilisin S11 and S12, 20 mg of lyophilized protein was added to 5 ml of 10 mM EDTA, 10 mM of tris (hydroxymethyl) amino-methane hydrochloric acid (hereinafter tris-HCl). Dissolved in pH 7.5 and dialyzed at 25 ° C. for 24 hours on the same buffer. To remove EDTA binding to subtilisin at low ionic strength, the protein was added twice for a total of 24 hours for 2 liters of 0.9 M NaCl, 10 mM Tris-HCl, pH 7.5, and then 2 liters of 2.5 mM Dialysis was performed three times for 24 hours at 4 ° C. against Tris-HCl, pH 7.5. Chelex 100 was added to all buffers containing no EDTA. When the C221 version without stabilizing amino acid substitutions was used, up to 50% of the protein precipitated during this process. It is essential that the titration experiments use pure natural apoenzymes so that the addition of calcium does not interfere with measuring the heat generated during binding when the calcium is added.

To ensure that the preparation of apo-subtilisin was not contaminated with calcium or EDTA, the samples were examined by titration with calcium in the presence of quin 2 and then titration calorimetry was performed.

Titration Calorimetry: Calorimetry Titration was performed using a microcal omega titration calorimetry, as described in detail in Wiseman et al., Analytical Biochemistry , 179: 131-137 (1989). Titration calorimetry consists of a matched reference cell containing a buffer solution and a solution cell containing a protein solution (1.374 ml). A microliter of liquor solution is added to the solution via a rotating stirrer syringe operated by a plunger driven by a stepping motor. After a stable background was achieved at a given temperature, automatic scanning was started and the accompanying heat change per scan was measured by a heat-coupled sensor between the cells. A sharp exothermic peak appeared during each injection, which returned to baseline before the next injection, which occurred 4 minutes later. The area of each peak represents the amount of heat involved in binding the added ligand to the protein. The total calories (Q) is then fixed to the total ligand concentration, [Ca] _total by the nonlinear least square minimization method [Wiseman et al., Analytical Biochemistry , 179: 131-137 (1989)] according to the following formula:

dQ / d [Ca] _total = ΔH [1/2 + (1- (1 + r) / 2-Xr / 2) / Xr-2Xr (1-r) + 1 + r ² ) ^1/2 ]

In the above formula, 1 / r is [P] _total × Ka, and X _r is [Ca] _total / [P] _total .

Binding of calcium to S11 and S12 subtilisin was determined by titration calorimetry, which allowed binding constants and binding enthalpy to be measured [Wiseman et al., Analytical Biochemistry , 179: 131-137 (1989); Schwarz et al., J. Biol. Chem. , 66: 24344-24350 (1991).

S11 and S12 subtilisin mutants were used in titration experiments because the production of wild type enzymes was not possible due to its proteolytic activity and low stability. Titration of S11 and S12 was performed at protein concentration [P] = 30 μM and 100 μM. Titration of S11 apoenzyme with calcium at 25 ° C. is shown in FIG. 4. The data points correspond to the negative column of calcium bonds associated with each titration of calcium. The titration calorimeter is sensitive to changes in K _a under conditions where the product of K _a × [P] is between 1 and 100 [Wiseman et al., Anaiytical Biochemistry , 179: 131-137 (1989)]. Since K _a for subtilisin is about 1 × 10 ⁷ M ⁻¹ , these protein concentrations result in values of K _a × [P] = 300 and 1000. At lower protein concentrations, the amount of heat generated during titration is difficult to measure accurately.

The results of fixing the titrations of S11 and S12 to the calculated curve are summarized in Table 6 below. Parameters in the table are binding parameters (n), binding constants (K _a ) and binding enthalpy (ΔH) for stoichiometric ratios. These parameters were determined from deconvolution using the nonlinear least square minimization method (Wiseman et al., Analytical Biochemistry , 179: 131-137 (1989)). The measurement for each experimental condition was performed in duplicate at 25 degreeC. The protein concentration was in the range of 30-100 μM, while the concentration of calcium solution was about 20 times the protein concentration. Each binding constant and enthalpy were based on several titration runs at different concentrations. Titration runs were performed until the titration peaks approached the background.

Determination of the appropriate calorie of the calcium A site of subtilisin mutants S11 and S12 Mutant [P] Parameters calculated from feet n Ka △ H S11 100 μM 0.98 ± 0.01 7.8 ± 0.2 x 10 ⁶ -11.3 ± 0.1 S11 33 μM 0.9 ± 0.3 6.8 ± 1.5 x 10 ⁶ -10.9 ± 0.2 S12 100 μM 0.99 ± 0.01 6.4 ± 0.2 x 10 ⁶ -11.8 ± 0.5

The average value obtained is similar for S11 and S12: ΔH = ˜-11 kcal / mol; K _a = 7 × 10 ⁶ M ⁻¹ and the stoichiometry of the bond was one calcium site per molecule. Weak binding site B does not bind calcium at concentrations below the millimolar range and therefore does not interfere with the measurement of binding to binding site A. The standard free energy of bonding at 25 ° C. is 9.3 kcal / mol. Therefore, the binding of calcium is primarily driven enthalpy, and the overall loss of entropy is small (ΔS _binding = -6.7 cal / ° mol).

(Example 3)

In vitro refolding of S15 subtilisin: Subtilisin was maintained at a concentration of approximately 100 μM as the original solution in 2.5 mM Tris-HCl pH 7.5 and 50 mM KCl for the refolding study. Proteins were denatured by diluting the original solution with 5 M guanidine hydrochloride (Gu-HCl), pH 7.5 or in most cases 25 mM H ₃ PO ₄ or HCl, pH 1.8-2.0. The final concentration of protein was 0.1-5 μM. S15 was completely denatured by these conditions within 30 seconds. S12 required approximately 60 seconds to fully denature. The acid-modified protein was then neutralized to pH 7.5 by the addition of tris-salt (if modified in HCl) or 5 M NaOH (if modified in H ₃ PO ₄ ). Refolding was initiated by adding KCl, NaCl, or CaCl ₂ to the desired concentration. For example, KCl was added with rapid stirring from 4 M original solution to a final concentration of 0.1-1.5 M. In most cases the restoration was performed at 25. The rate of reconstruction is from an increase in absorbance at λ = 286, from an increase in intrinsic tyrosine and tryptophan fluorescence (absorbance λ = 282, luminescence λ = 347) by μυ absorbance, or at a negative ellipticity at λ = 222 nm Spectroscopically measured by circular dichroism from increase.

(Example 4)

X-ray crystallography: Large single crystal growth and X-ray diffraction data collection were performed essentially as reported in the literature [Bryan et al., Proteins: Struct. Funct. Genet. , 1: 326-334 (1986b); Pantoliano et al., Biochemistry , 27: 8311-8317 91988); Pantoliano et al., Biochemistry , 28: 7205-7213 (1989). However, it is not necessary to deactivate the S221C variant with diisopropyl fluorophosphate (DFP) in order to obtain a suitable crystal. The starting model for S12 was made from ultrastable subtilisin mutant 8350 (protein data bank entry 1SO1.pdb). The S12 structure was purified and then modified to serve as the starting model for S15.

Two models were refined using a limited minimum-square technique using a data set with about 20,000 reflections between 8.0 and 1.8 Å resolution [Hendrickson et al., “Computerization of Crystallography” in Diamond et al., eds., Bangalore: Indian Institute of Science , 13.01-13.23 (1980). The initial differential map for S15 was imaged by a version of S12 with the entire region A region omitted, which showed a continuous density that clearly represents an uninterrupted spiral, which allows the initial S15 model to be constructed and purification to begin. Let's do it. Each mutant was purified in approximately 80 cycles from R of approximately 0.30 to R of approximately 0.18, and the calculation of the electron density map and graphics modeling program FRODO [Jones, J. Appl. Crystallogr. , 11: 268-272 (1978).

With the exception of the deleted calcium-binding loop region, the structures of S12 and S15 are very similar and the root-square mean (r.m.s.) deviation is 0.18 mm 3 between 262 α-carbons. The N-terminus of S12 is next to the Site A loop (as in the wild type) and provides one calcium coordination ligand, the side chain oxygen of Q2. In S15 the loop disappears and residues 1 to 4 become disordered. In S12 the site A loop (as in the wild type) is present as an obstruction at the last turn of the alpha helix of 14 residues; In S15, this helix is unobstructed and represents a normal helix geometry over its entire length. Diffusion differential density and higher temperature factors indicate that there is some disorder in the newly exposed residues around the deletion.

(Example 5)

Differential Scanning Calorimetry: The stability properties of S12 and S15 were studied using differential scanning calorimetry (DSC). The Δ75-83 mutant (S15) is very similar at the melting point temperature to the apoenzyme of S12. DSC profiles of Apo-S12 and S15 are shown in FIG. 3. The maximum heat capacity temperature is 63.0 ° C. for S15 and 63.5 ° C. for Apo-S12 at pH 9.63. DSC experiments were performed at high pH to avoid aggregation during denaturation. The amount of excess heat absorbed by the protein sample increased through the transition from the folded state to the unfolded state at a constant pressure as temperature, which enabled a direct measurement of ΔH of unfolding [Privalov et al., Methods Enzymol. 131: 4-51 (1986)]. ΔH _cal of the spread for Apo-12 and S15 is about 140 kcal / mol. Above pH 10.0, the unfolding transition for S15 fits well with the two-state model and is consistent with the equilibrium thermodynamics as indicated by the Vandt Hof equation (dln K / dT = _{ΔH vH} / RT ² ). That is, _{ΔH v} H (bart hop enthalpy or apparent enthalpy) is approximately equal to _{ΔH cal} (calorimeter or realistic enthalpy). However, at pH 9.63, the melt profile for the two proteins is asymmetric, indicating that unfolding is not a pure two-phase process.

(Example 6)

Measurement Dynamics of Calcium Separation: Separation of calcium from subtilisin is a slow process. Queen 2, a fluorescent calcium chelator, was used to measure this rate. Quine 2 binds to calcium with _a K _a value of 1.8 × 10 ⁸ at pH 7.5 [Linse et al., Biochemistry , 26: 6723-6735 (1987)]. Fluorescence of Quin 2 at 495 nm increases approximately 6-fold when bound to calcium [Bryant, Biochem. J. , 226: 613-616 (1985). The subtilisin S11 or S12 in the isolated state contains one calcium ion per molecule. The kinetics of calcium release from the protein when mixed with excess Quin 2 can be traced from the increase in fluorescence at 495 nm. The reaction is assumed to follow the path N (Ca) ⇔N + Ca + Queen 2⇔Queen (Ca). Separation of calcium from subtilisin is very slow compared to calcium binding by quin 2, so the change in fluorescence of quin 2 is equal to the rate at which calcium is separated from subtilisin. As can be seen in FIG. 5A, the initial release of calcium from S11 follows simple primary dynamics.

Temperature dependence of calcium separation: The primary rate constant (k) for calcium separation was measured from 20 ° to 45 ° C. The plot of ln k versus 1 / T ° K is approximately linear. Calcium separation data were curve fit using the transition state theory following the Err equation:

△ G ^‡ = -RT ln K ^‡ = -RT ln kh / k _B T

Where k _B is the Boltzmann constant, h is the flank constant, and k is the primary rate constant for folding. A graph of ln hk / k _B T vs 1 / T is shown in FIG. 5B.

The data was then curve fitted according to the following equation [Chen et al., Biochemistry , 28: 691-699 (1989)]:

ln K ^‡ = A + B (To / T) + C ln (To / T)

Wherein A = [ΔCp ^‡ + ΔS ^‡ (To)] / R; B = A-ΔG ^• (To) / RTo; C = △ Cp ^‡ / R. The data obtained give the following results: DELTA G ^• = 22.7 kcal / mol; ΔCp ′ ^‡ = −0.2 kcal / ° mol; ΔS ′ ^‡ = − 10 cal / ° mol; And ΔH ' ^‡ = 19.7 kcal / mol reference temperature, 25 ° C. Some possible curvature of the plot may be due to changes in heat capacity associated with the formation of the transition state (ΔCp ' ^‡ = 0.2 kcal / ° mol). ΔCp for protein folding was closely correlated with changes in the exposure of hydrophobic groups to water [privalov et al., Adv. Protein Chem. 39: 191-234 (1988); Livingstone et al., Biochemistry , 30: 4237-4244 (1991). Therefore, in terms of heat capacity, the transition state appears to be similar to natural protein. The values for ^ΔS ′ ^‡ and ΔH ′ ^‡ obtained from FIG. 5B indicate that the transition state is enthalpy less desirable than the calcium bound state showing only a small change in entropy.

Other embodiments of the invention will be apparent to those skilled in the art upon consideration of the specification of the invention and the invention set forth therein. It is to be understood that the specification and examples are exemplary only, with a true spirit and scope of the invention being indicated by the following claims.

(Sequence list)

(1) General Information

(i) Applicant: Brian, Philip N

Alexander, Patrick

Straussberg, Susan L

(ii) Name of the Invention: Calcium-Unbound Subtilisin Mutant

(iii) Number of sequences: 1

(iv) Correspondence address:

(A) To: Doanne, Skunker & Mathis

(B) Distance: P. Box: 1404

(C) Poetry: Alexandria

(D) State: Virginia

(E) Country name: United States of America

(F) Zip code: 22313-1404

(v) Computer-readable form:

(A) Media Type: Floppy Disk

(B) Computer: IBM PC Compatibility

(C) operating system: PC-DOS / MS-DOS

(D) Software: Patterned Release # 1.0 Version # 1.25

(vi) pending application data:

(A) Application number: WO PCT / US95 / 05520

(B) Application date: April 28, 1995

(C) Classification code:

(vii) First application data:

(A) Application number: US 08 / 309,069

(B) Application date: September 20, 1994

(viii) Representative Information:

(A) Name: Mutsu, Donna M

(B) Registration Number: 36,607

(C) Case Number: 028758-003

(ix) Telecommunication information:

(A) Telephone: (703) 836-6620

(B) Fax: (703) 836-2021

(2) Information about SEQ ID NO: 1 (SEQ ID NO: 1)

(i) Sequence features:

(A) Length: 1868 base pairs

(B) Type: nucleic acid

(C) strands: single

(D) mode: linear

(ii) Molecular type: DNA (genome)

(ix) characteristics:

(A) Name / Key: CDS

(B) Location: 450..1599

(ix) characteristics:

(A) Name / Key: mat_ peptide

(B) Location: 772..1599

(ix) characteristics:

(A) Name / Key: misc_character

(B) location: 450

(C) Other information: / week = "Amino acid Val at position 450 is fMet."

(xi) SEQ ID NO: SEQ ID NO: 1: (SEQ ID NO: 1)

[SEQ ID NO: 1a]

[SEQ ID NO: 1b]

Claims (15)

An enzymatically active subtilisin protein mutated to remove the calcium binding ability of the subtilisin protein at the high affinity calcium binding site, wherein the mutated subtilisin protein comprises a deletion of amino acids at positions 75-83. Characterized by a subtilisin mutant. The method of claim 1, wherein the subtilisin mutant is at least one of N218S, M50F, Y217K, P5S, D41A, K43R, K43N, Q271E, Q2K, Q2W, Q2L, A73L, A73Q, Q206C, Q206V, Q206I, Q206W or S3C. Subtilisin mutant, characterized in that it further comprises a substitution mutation. 3. The subtilisin mutant of claim 2, wherein the mutant has one or more additional mutations in β-ribbon amino acids 202-219. 3. The subtilisin mutant of claim 2, wherein the subtilisin mutant comprises substitutional mutations of N218S, M50F, Y217K and P5S. 3. The subtilisin mutant of claim 2, wherein the subtilisin mutant comprises substitutional mutations of N218S, M50F, Y217K, Q271E, Q2K, A73L and Q206V. 3. The subtilisin mutant of claim 2, wherein the subtilisin mutant comprises substitutional mutations of N218S, M50F, Y217K, Q271E, Q2K, A73L and Q206C. 3. The subtilisin mutant of claim 2, wherein the subtilisin mutant comprises substitutional mutations of N218S, M50F, Y217K, Q271E, Q2K, A73L, Q206C and S3C. The subtilisin mutant of claim 1, wherein the subtilisin is derived from a Bacillus strain. 9. The subtilisin mutant of claim 8, wherein the subtilisin mutant is a subtilisin BPN 'mutant, a subtilisin Carlsberg mutant, a subtilisin DY mutant, a subtilisin amylosacariticus mutant or a subtilisin mesentico A subtilisin mutant, characterized in that it is a peptidase mutant or a subtilisin sabinase mutant. 10. The subtilisin mutant of claim 9, wherein the subtilisin mutant is a subtilisin BPN 'mutant. The high affinity calcium binding site is mutated to remove the calcium binding ability of the subtilisin protein, and the mutated subtilisin protein is modified at amino acids 75-83 corresponding to nucleotide positions 993-1019 of SEQ ID NO: 1 A recombinant method for providing expression of an enzymatically active subtilisin protein comprising a deletion, the method comprising the following steps: (a) transforming the recombinant host cell with an expression vector containing enzymatically active subtilisin DNA that provides expression of subtilisin that does not bind calcium protein upon expression; (b) culturing the host cell under conditions that will provide expression of an enzymatically active subtilisin mutant; And (c) recovering the enzymatically active subtilisin mutant expressed from said microbial host. The method of claim 11, wherein the subtilisin mutant is at least one of N218S, M50F, Y217K, P5S, D41A, K43R, K43N, Q271E, Q2K, Q2W, Q2L, A73L, A73Q, Q206C, Q206V, Q206I, Q206W or S3C. Recombinant method characterized in that it further comprises a substitution mutation. 13. The method of claim 12, wherein the subtilisin mutant is a subtilisin BPN 'mutant. The high affinity calcium binding site is mutated to remove the calcium binding capacity of the subtilisin protein, and the mutated subtilisin protein is modified at amino acids 75-83 corresponding to nucleotide positions 993-1019 of SEQ ID NO: 1. A recombinant DNA comprising a deletion, wherein the mutated subtilisin protein encodes a subtilisin protein that retains enzymatic activity and stability. 15. The method of claim 14, wherein the subtilisin DNA is at least one of N218S, M50F, Y217K, P5S, D41A, K43R, K43N, Q271E, Q2K, Q2W, Q2L, A73L, A73Q, Q206C, Q206V, Q206I, Q206W or S3C. Recombinant DNA, characterized in that the subtilisin BPN 'coding sequence further comprising a substitution mutation.

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